【摘要】：古紫質(zhì)-4(aR4)是新近發(fā)現(xiàn)的古生菌Halobacterium speciesxz515菌紅膜上的唯一的光敏視黃醛蛋白,和細(xì)菌視紫紅質(zhì)(bR)具有相似的質(zhì)子泵功能,但在中性pH條件下,兩者的質(zhì)子釋放和攝取順序相反。深入研究質(zhì)子供體天冬氨酸97(Aspartic acid 97,D97)對蛋白光循環(huán)、質(zhì)子泵功能和能量轉(zhuǎn)化效率影響的作用機理,并從分子水平上揭示aR4質(zhì)子釋放和攝取時序反轉(zhuǎn)內(nèi)在原因具有重大的指導(dǎo)意義。研究工作采用基因定點突變技術(shù),在宿主細(xì)胞Halobacterium speciesL33中成功表達(dá)出了 aR4的單突變體D97N,以及相對應(yīng)的bR單突變體D96N。采用固體核磁共振技術(shù)、紫外-可見吸收光譜、閃光動力學(xué)光譜和低溫透射紅外光譜等譜學(xué)手段,對蛋白視黃醛鍵合區(qū)、光反應(yīng)中間態(tài)、質(zhì)子泵功能以及ATP生成率等進(jìn)行了原生膜環(huán)境條件下的原位表征。閃光動力學(xué)光譜測定結(jié)果表明,D97突變可造成aR4的M態(tài)衰減時間延長至秒級,并使蛋白喪失質(zhì)子泵功能,而對應(yīng)的D96N突變則造成了 bR的M態(tài)衰減時間延長和O態(tài)的消失,但是蛋白仍具有質(zhì)子泵功能。固體核磁共振、紫外-可見光譜、蛋白聚集狀態(tài)及其ATP生成率等數(shù)據(jù)顯示,不論是aR4中的D97還是bR中的D96,都距視黃醛發(fā)色團(tuán)有10A之遠(yuǎn),突變沒有對視黃醛鍵合區(qū)產(chǎn)生顯著的影響,但對質(zhì)子泵功能和蛋白的三聚結(jié)構(gòu)產(chǎn)生了一定的影響,使得突變體菌株ATP的生成率下降了 50%。低溫透射紅外光譜測定結(jié)果表明,與bR中的D85不同,aR4中的D86質(zhì)子化過程提前到了L態(tài),并一直維持到了N態(tài)。所有研究結(jié)果揭示,同為質(zhì)子供體的D97和D96在aR4和bR光循環(huán)中發(fā)揮著不同的作用,表明aR4與bR存在著不同的光循環(huán)中間態(tài),而這與二者質(zhì)子釋放和攝取時序反轉(zhuǎn)有著直接的對應(yīng)關(guān)系。
[Abstract]:Paleoviolin-4 (aR4) is the only Guang Min retinaldehyde protein on the erythrocyte membrane of Halobacterium speciesxz515, which has similar proton pump function to bacteriorhodopsin (bR), but under neutral pH condition. The order of proton release and uptake is opposite. The effects of proton donor aspartic acid 97 (Aspartic acid 97 / D97 on protein photocycling, proton pump function and energy conversion efficiency were studied. It is of great significance to reveal the internal causes of proton release and uptake time reversal of aR4 at molecular level. The single aR4 mutant D97N and the corresponding bR single mutant D96N were successfully expressed in host cell Halobacterium speciesL33 by site-directed mutation. By means of solid-state nuclear magnetic resonance spectroscopy, UV-Vis absorption spectrum, flash kinetic spectroscopy and low-temperature transmission infrared spectroscopy, the binding region of protein retinaldehyde and the intermediate state of photoreaction were studied. The proton pump function and ATP formation rate were characterized in situ under the condition of primary membrane environment. The results of flash kinetic spectroscopy show that D97 mutation can prolong the M state decay time of aR4 to the second order and make the protein lose the proton pump function. The corresponding D96N mutation results in the prolongation of M state decay time and the disappearance of O state in bR. But protein still has proton pump function. Solid nuclear magnetic resonance (NMR), UV-Vis spectroscopy, protein aggregation state and their ATP production rate showed that both D97 in aR4 and D96 in bR were 10 A away from the retinol chromophore. The mutation had no significant effect on the retinaldehyde binding region, but had a certain effect on the proton pump function and the trimeric structure of the protein, resulting in a 50% decrease in the ATP production rate of the mutant strain. The results of low temperature transmission infrared spectroscopy show that, unlike D85 in bR, the protonation of D86 in aR4 advances to L state and maintains to N state. All the results show that D97 and D96, which are the same proton donors, play different roles in the optical cycle of aR4 and bR, indicating that there are different intermediate states in the optical cycle between aR4 and bR. This has a direct relationship with proton release and uptake time reversal.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q51

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本文編號：2417370